Antitumor-Promoting and Anti-inflammatory Activities of Licorice

Chapter 31

Antitumor-Promoting and Anti-inflammatory Activities of Licorice Principles and Their Modified Compounds

Downloaded by PENNSYLVANIA STATE UNIV on July 29, 2012 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch031

Shoji Shibata Shibata Laboratory of Natural Medicinal Materials, 3rd Tomizawa Building, 4th Floor, Yotsuya 3-2-7, Shinjuku-ku, Tokyo 160, Japan

Glycyrrhetinic acid, the aglycone of the licorice saponin glycyr rhizin, showed anti-tumor promoting effects in vitro and in vivo in the two-stage tumorigenic experiment using D M B A / T P A . Based on this finding, 18β- or 18α-olean-12-ene-3β,28-diol and 3β,23,28-triοl and their hemiphthalates, prepared from oleanolic acid and hederagenin, were tested and found to possess potent antitumor promoting activities. Licochalcone A from Xin-jiang licorice showed remark able inhibitory activities against DMBA/TPA-induced tumorigenesis and TPA-induced inflammatory edema of mice, which suggests an intimate correlation between antitumorigenic and antiinflammatory mechanisms.

Licorice is the sweet tasting root of various species of Glycyrrhiza (Leguminosae) which has been used in the East and West since ancient times as a medicine and a sweetening agent. In Dioscorides' De Materia Medica, licorice was described as an antiinflammatory drug for pharyngitis, and in old Chinese medical books it was described as being an antispasmodic agent as well as being a harmonizer to soften various drug actions. Therefore, licorice has frequently been combined with other herb drugs in the numerous prescriptions commonly used in traditional Chinese medicine. On the other hand, licorice is widely employed as a food sweetener. Various species of licorice are sold on market under various trade names according to their habitat. Their botanical origins have been identified. Some species of Glycyrrhiza are not used for medicinal purpose or as food additives owing to their bitter taste (Table I). Licorice's Saponin Component, Glycyrrhizin A l l the licorice species used medicinally contain a sweet tasting triterpenoid saponin, glycyrrhizin (GL), at a fairly high level (average: 4-5% of dry wt.) as the main principle. Some satellite triterpenoid saponins and various types of flavonoids are also found in licorice at a much lower level.

0097-6156/94/0547-0308$06.00/0 © 1994 American Chemical Society

In Food Phytochemicals for Cancer Prevention II; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

31. SHIBATA

309

Antitumor-Promoting Activities of Licorice Table I. Source Plants of Licorice for Medicinal Use

Plant

Location

Glycyrrhiza uralenesis Fisch.

Don-bei, Xi-bei, Xin-jiang (China)

Gl. glabra L .

Xin-jiang, Turkumenistan Azerbaijan, Afganistan Iran, Iraq, Turkey


var. glandulifera Rgl. et Herd var. pubescens Litw.

Xin-jiang

Gl. inflata Batal Gl. korshinskyi G. Hrig. Gl. aspera Pall.

"

Gl. echinata L. and Gl. pallidiflora Maxim, have no medicinal use

G L is a β(1—>2) linked glucuronyl glucuronide of glycyrrhetinic acid (GA, 18P-olean-ll-one-12-ene-3P-ol-30-oic acid). The stereochemical β-linkage of glu curonyl moiety at the 3p-hydroxyl of G A has often been wrongly referred to as a, as noted in the Merck Index (7), based on the classical data of molecular rotation (2) . In truth, it is β on the basis of C N M R (103.3 ppm, C - l ' ) and H N M R (4.31 ppm H d, J=6.9 Hz; 4.47 ppm 1H d, J=7.3 Hz) data obtained by Russian workers (3) and by us (Shibata, S.; Iwata, S., unpublished data, 1991) (Figure 1, Table II). Kitagawa and his group have intensively investigated the minor satellite saponins in the roots of Gl. uralensis and Gl. inflata to determine their chemical structures (4-6) (Table III). A l l of these have β-linkages with their sugar moieties. 1 3

l

l

Biological Activities of Glycyrrhizin and Glycyrrhetinic Acid The biochemical and pharmacological activities of G L and G A have been investi gated by several workers. More distinct in vivo biological activities have been demonstrated by G L than by G A , owing probably to its water-solubility and higher absorbability; nevertheless, that does not exclude the basic biological activity of G A, the aglycone. Clinical. The well-recognized pharmacological effects of licorice are the anti inflammatory (7) and antiallergic effects (8) of the principal component, G L . Recently, interferon inducing activity (9) and an antiviral effect against HSV (10) by G L has been demonstrated clinically. Several preparations of G L and G A are now available for clinical use, but side effects, such as pseudo-aldosteronism causing edema, hypertension, and hypopotassemia in patients during long term and high dosage administration, should be noted. Strong Neo Minophagen C (SNMC, a GL-preparation combined with L-cysteine and glycine) for i.v. injection and Glycyron Tablet (GL combined with L-methionine and glycine) for p.o. administration are available in Japan. The amino acids are added to reduce the pseudo-aldosteronism of GL. These GL-preparations, which have originally been used as an antiallergic drug, are clinically employed against viral chronic hepatitis (77) and recently, applied to HIV-carriers to prevent


FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS

310

Table II.

1 3

C - N M R Spectrum of Glycyrrhizin


Glycyrrhetinic acid moiety C-3 C-ll C-12 C-13 C-18 C-30

Glucuronic acid moiety c-r C-2' C-3' C-4' C-5' C-6' c-r C-2" C-3" C-4" C-5" C-6"

88.1 ppm 198.8 127.2 169.5 47.9 177.5

103.3 ppm 82.5 75.0 71.1 75.7 170.0 104.6 74.5 75.5 71.4 76.1 169.8

H-NMR signal of anomeric protons 4.31 ppm ( H d, J = 6.9 Hz) 4.47 ppm (*H d, J = 7.3 Hz) l

OH Glycyrrhizin (GL)

Figure 1. Structures of glycyrrhetinic acid (GA) and glycyrrhizin (GL).



2

2

2

2

3

3

2

3

2

2

glycyrrhetinic acid (GA) GA deoxo-GA deoxo-GA-11,13-diene deoxo-GA GA-22-lactone GA-22-lactone GA-24-OH liquiritic acid (30oc-GA) deoxo-GA-24-OH deoxo-GA-11,13-diene-24-OH deoxo-GA-24-OH GA GA uralenic acid (18oc-GA)

glycyrrhizin (GL) licorice saponin (LS)-A3 LS-B LS-C LS-D LS-E LS-F LS-G LS-H LS-J LS-K LS-L arabo-GL apio-GL 18oc-GL

GL GL GL GL GL GL GL GL GL GL GL GL GL GL GL

uralensis, GL inflata uralensis, GL inflata uralensis uralensis uralensis uralensis uralensis uralensis, GL inflata uralensis, GL inflata uralensis uralensis uralensis (Xin-jiang) inflata inflata inflata

Sapogenin

Saponins

Source

Table III. Oleanane-type Triterpenoid Saponins of Licorice

GlcA(P2->l)GlcA GlcA-GlcA(30)GlcA GlcA-GlcA GlcA-GlcA GlcA-GlcA(p2->l)Rha GlcA-GlcA GlcA-GlcA-Rha GlcA-GlcA GlcA-GlcA GlcA-GlcA GlcA-GlcA GlcA(P2->l)Ara(p2->l)Rha GlcA(p2->l)Ara GlcA(p2->l)Api GlcA-GlcA

Sugar moiety


312


the progression of infection (Ishida, N . ; Shigeta, S., personal communication, 1992). Carbenoxolone sodium, hemisuccinate of GA, has been developed in Britain for the treatment of peptic ulcer (72). The pseudo-aldosteronism of G L and G A has been explained by the com petitive binding of G A to the renal glucocorticoid receptor (13) and inhibition by G A of A 5p-steroid reductase (14,15) and Na K -ATPase associated with the Na -extrusion pump (16). The inhibition by G A of 11 β-hydroxysteroid dehydrogen ase in skin (17) and lung tissues (Schleimer, R.P., personal communication, 1991) resulting in retention of endogenous Cortisol may explain the clinical anti inflammatory action of GL, GA, and their preparations. 4

+

+


+

In Vitro. Inhibition of several distinct biological and biochemical functions has been demonstrated in vitro by G A in such as intercellular gap juncture communi cation (18,19), phosphotransferase activity of protein kinase C (20), phospholipid metabolism promoted by 12-0-tetradecanoylphorbol-13-acetate (TPA) in HeLa cells (27), and 3-O-methylglucose transport stimulated by TPA (22). Biological activities of G L on enzymatic actions and on cultured cells and organs are mostly demonstrated by the aglycone form, GA, but G A has fairly high cell-toxicity against host cells. Therefore, for in vitro experiments on antiviral action, G L is employed (23-28). Antiviral activities of G L were shown in vitro against HSV-1, New Castle disease, Vescular stomatitis, and Polio-1 viruses (23, 24). Inhibition of proliferation of Varicella Zoster virus (27) and HIV-1 (28) by G L were also demonstrated. The antiviral effect against HIV-1 is attributed to the inhibition of adsorption of virus on the surface of lymphocytes, but not to the inhibition of viral RNA-reverse tran scriptase. Antitumor Promoting Effects of Glycyrrhetinic Acid In the course of carcinogenesis, there are at least two stages, initiation and promotion (29). The promotion stage would be followed by progression esta blishing malignant state of cancer. The best established experimental model is the two-stage mouse skin carcinogenesis system. The initiator, 7,12-dimethylbenz[a]anthracene (DMBA), is applied to the back skin of mice in a single dose that is under the threshold dosage for carcinogenesis. After some interval, the promoter TPA — an irritating principle of croton oil isolated from the seeds of Croton tiglium — is applied to the back by painting repeatedly for 18-20 weeks. TPA is effective at very low concentrations (10~ -10~ M), and the formation of papillomas has been demonstrated within a few weeks. Other irritant products, such as teleocidin, aplysiatoxin, and palytoxin have also been found to be effective cancer promoters for DMBA-initiated mice (30). These two-stage carcinogenesis systems are useful for the survey of anti tumor promoters and have been used to study cancers and organs other than skin papillomas induced by the D M B A / T P A system. But the in vivo experiments performed by either topical or oral administration of inhibitors require a long time to evaluate their potencies. More convenient, short-term, experimental systems have been developed as screening tests. For example, an in vitro experiment measuring the inhibitory potency of test compounds against TPA-promoted phospholipid metabolism in cultured cells is employed (31,32). 9

8


31. SHIBATA

Antitumor-Promoting Activities ofLicorice

313

Since the tumor promoting agents are mostly skin irritating compounds, antiinflammatory agents have been tested for the antitumor promoting activities. As antiinflammatory agents occurring in nature, G L and G A were tested for inhibition of TPA-promoted phospholipid metabolism. Incorporation of P i into phospho lipids of HeLa cells was increased 4-fold by 50 n M TPA. G A (100 μΜ) inhibited the increase by 50%. In this experiment, G L was not significantly effective (32). The result of in vitro screening tests prompted us to investigate the effect of G A on in vivo two-stage carcinogenesis. The in vivo test was carried out as follows: The backs of 7-week-old ICR-mice (15 mice per group) were shaved with an electric clipper. After 2 days, a single application of 100 μg D M B A was applied to the shaved area. T P A (0.5 μg, 0.81 nmol) was applied by painting twice a week starting 1 week after initiation. G A (10 μΜ) was dissolved in 100 μΐ DMSO: acetone (1:99) and applied topically 40 min before each T P A application. The number and sizes of the skin tumors induced were determined once a week for 18 weeks. The percentage of tumor bearing mice in the group treated with G A was 40% at week 20 in contrast to 97% in the control. The average number of tumors per mouse in the D M B A / T P A alone group was 9.6 at week 20, whereas that in the D M B A / T P A plus 10 μΜ G A group was 1.8 (32). Antitumor promoting activity has now been added to the multifunctional biological activities of GA, the sapogenin of GL. When tumor promoters do not follow initiation, they merely act as inflammatory agents. Therefore, the anti inflammatory activities against TPA-induced mouse ear edema and those induced by other agents have been used as screens for antitumor promoting effects. Destruction of α,β-unsaturated carbonyl at the 11-position and reduction of carboxyl attached at the 20-position of G A produced 18p-olean-12-ene-3p, 30-diol (deoxoglycyrrhetol), 18p-olean-9(ll)12-diene-3P,30-diol (homoannular diene), and olean-ll,13(18)-diene-3β,30-diol(heteroannular diene) (Figure 2). These com pounds and their hemiphthalates, prepared by several chemical reaction steps, have inhibitory activity against lipoxygenase and cyclooxygenase, showing their anti inflammatory actions. Actually, by topical and oral administration of the dihemiphthalates or the sodium salts of these GA-modified compounds showed dose-dependent inhibitory actions against TPA-induced and arachidonic acid (AA)-induced mouse ear edemata (33-35).


32

Antitumor Promoting Effects of Oleanane-type Triterpenoid Compounds Prepared from Oleanolic Acid and Hederagenin The fact that G A showed antitumor promoting effects in the two-stage tumori genesis system revealed a possibility that such a biological activity might be shared by other members of naturally occurring triterpenoids. Thus, oleanolic acid, hedera genin, ursolic acid, echinocystic acid, entagenic acid, and saikogenin A were tested in the same assay system. Oleanolic acid and hederagenin, which are the most abundant triterpenes in natural sources except for glycyrrhetinic acid, were chemically modified in their functional groups, modulating their biological effects. The 28-COOH group was converted to C H O H , and 18β-Η partly converted to 18oc-H to yield 18β- or 18oc-olean-12-ene-3P,28-diol from oleanolic acid and 18β- or 18a-olean-12-ene3β,23,28-ίπο1 from hederagenin (Figure 3). The enhancement of biological activi ties by these functional and stereochemical conversions has been demonstrated in GA. 2


In Food Phytochemicals for Cancer Prevention II; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994. 2

2

Deoxoglycyrrhetol Heteroannular diene (18- β-olean-l 2-ene-3 (3,30-diol) Figure 2. Chemical modification of glycyrrhetinic acid (GA), showing the destruction of an α,β-unsaturated carbonyl system in the C ring and conversion of -COOH at C-20 into CH2OH. The therapeutic activities enhanced are anti inflammatory, antiallergic and antiulcerous. Vitride: NaAlH (OCH3CH2CH OCH3)2


31.

SHIBATA

315

Antitumor-Promoting Activities of Licorice

OH


COOR' Vitridg

HO' 23R R = CH 18p-Erythrodiol m.p. 236-237°, [ a]D +77° (Chf) R = CH OH 18 β-Olean-l 2-ene-3 p,23,28-triol m.p. 216-217°, [ oc]D +78.4° (Chf)

HO'

N

R = CH 3, R = H Ιββ-Oleanolic acid R = CH OH, R ' = H 18 β-Hederagenin

3

2

2

Cr0 (tBu) in AcOH 60° 4 hrs 2

2

NaOH in COOH

COOH

RO

Vitride in THF

CH OHp / H 28 — * 2

d

C

2

CH OH 2

HO 23 R R = CH 18a-Olean-12-ene-3 p,28-diol m.p. 264-266°, [ oc]D +41 ° (Chf) R = CH OH 18 a-Olean-12-ene-3 p,23,28-triol m.p. 258-263°, [ a]D +56° (Chf) N

3

2

Figure 3. Structural modifications to oleanoic acid and hederagenin.


316


In Vitro Tests. Antitumor promoting activities of oleanane-type triterpenes were primarily screened by their inhibitory action against TPA-induced phospholipid metabolism by measuring Pi-incorporation into phospholipids of HeLa cells. The chemical and stereochemical conversion of functional structures of the triterpenoid compounds resulted in potentiation of their inhibitory effects as expected. 18a- and p-olean-12-ene-3P,23,28-triol also showed almost the same level of inhibition of Pj-incorporation using C3H10T 1/2 cells and Swiss 3T3 cells. 32


32

In Vivo Tests. Topical application of 18a- or 18P-olean-12-ene-3p,23,28-triol (81 nM) on the shaved back of mice treated with D M B A (100 μg) and TPA (0.5 μg, 0.81 nmol/mouse, twice a week) resulted in 80% or 60% inhibition, respectively, of tumor formation at week 18. The average number of tumors per mouse was 10.6 in the control group at week 18, whereas it was 0.6 (p

Antitumor-Promoting and Anti-inflammatory Activities of Licorice

Recommend Documents